1. Field of Invention
This invention relates generally to improved methods for growing various mammalian cells in vitro using cell culturing methods and novel cell culture surface compositions and methods of application.
2. Description of Prior Art
Cell transplantation has been proposed as an alternative for total organ replacement for a variety of therapeutic needs, including treatment of diseases in the eye, brain, liver, skin, cartilage, and blood vessels. See, for example, J P Vacanti et al., J. Pediat. Surg., Vol. 23, 1988, pp. 3-9; P Aebischer et al., Brain Res. Vol. 488, 1998, pp. 364-368; C B Weinberg and E. Bell, Science, Vol. 231, 1986 pp. 397-400; I V Yannas, Collagen III, M E Nimni, ed., CRC Press, Boca Raton, 1988; G L Bumgardner et al., Hepatology, Vol. 8, 1988, pp. 1158-1161; A M Sun et al., Appl. Bioch. Biotech., Vol. 10, 1984, pp. 87-99; A A Demetriou et al., Proc. Nat. Acad. Sci. USA, Vol. 83, 1986, pp. 7475-7479; W T Green Jr., Clin. Orth. Rel. Res., Vol 124. 1977, pp. 237-250; C A Vacanti et al., J. Plas. Reconstr. Surg., 1991; 88:753-9; P A Lucas et al., J. Biomed. Mat. Res., Vol. 24, 1990, pp. 901-911. The ability to create human cell lines in tissue culture will enhance the prospect of cell transplantation as a therapeutic mode to restore lost tissue function. It is especially vital to be able to create human cultured cell lines from tissues of the neural crest, since tissues or organs derived from that origin cannot usually repair itself from damage after an individual reaches adulthood.
Conventional tissue culture lab wares useful in growing cells in vitro, are usually coated with a negative charge to enhance the attachment and sometimes proliferation of mammalian cells in culture. However, traditionally it has been most difficult to achieve a satisfactory attachment, maintenance, and propagation of mammalian neuronal cells with the conventional tissue culture surfaces. Improvements have been made by adding layers of collagen gel or depositing an extracellular matrix secreted by rat EHS tumor cells onto the tissue culture plates and dishes to facilitate neural cell attachment and proliferation. These techniques, however, are hindered by the shortcoming that the material has to be layered on the culture surfaces shortly before the cells are seeded.
The use of a biopolymer carrier to support the attachment, growth, and eventually as a vehicle to carrying the cells during transplantation is vital to the success of cell replacement therapy, particularly in the brain and the back of the eye, where cells derived from the neural crest origin is often damaged during the aging process. There are seven general classes of biopolymers: polynucleotides, polyamides, polysaccharides, polyisoprenes, lignin, polyphosphate and polyhydroxyalkanoates. See for example, U.S. Pat. No. 6,495,152. Biopolymers range from collagen IV to polyorganosiloxane compositions in which the surface is embedded with carbon particles, or is treated with a primary amine and optional peptide, or is co-cured with a primary amine- or carboxyl-containing silane or siloxane, (U.S. Pat. No. 4,822,741), or for example, other modified collagens are known (U.S. Pat. No. 6,676,969) that comprise natural cartilage material which has been subjected to defatting and other treatment, leaving the collagen II material together with glycosaminoglycans, or alternatively fibers of purified collagen II may be mixed with glycosaminoglycans and any other required additives. Such additional additives may, for example, include chondronectin or anchorin II to assist attachment of the chrondocytes to the collagen II fibers and growth factors such as cartilage inducing factor (CIF), insulin-like growth factor (IGF) and transforming growth factor (TGFβ).
Until the advent of the present invention, it was not possible to culture mammalian or human neuronal tissues from the neural crest or individual neurons and get them to grow and divide in vitro.